The structural analysis of polynucleotides plays an increasingly important role in modern molecular biology. With the advent of polynucleotide amplification technology, e.g., PCR, and projects directed towards sequencing the human genome, the level of interest in this area is high. This heightened interest has lead to the need for analytical systems with increased resolution, throughput, and degree of automation.
Techniques for polynucleotide analysis typically involve treatment of a polynucleotide sample with specific enzymes wherein the enzyme treatment creates mixtures of variable-length polynucleotide fragments, followed by a size-based electrophoretic separation of the reaction products. Typically, in order to increase the throughput of the electrophoresis step, multiple separations are performed in parallel using a rectangular "slab" gel.
Modern polynucleotide analysis methods use fluorescence-based detection schemes to detect the electrophoretically separated products. These methods typically employ fluorescently labeled samples in combination with multicolor real-time multilane fluorescence detectors capable of interrogating the multiple lanes of a slab gel, e.g., Hunkapiller et al., U.S. Pat. No. 4,811,218, the disclosure of which is hereby incorporated by reference. Fluorescence-based detection schemes provide several advantages over classical radioactive methods: (i) multicolor fluorescent labeling systems allow multiple differentially labeled reaction products to be run in a single electrophoresis lane, thereby increasing throughput; (ii) fluorescence systems are better suited to modern high-performance electrophoresis formats, e.g., capillary electrophoresis; (iii) real-time fluorescence detectors provide data in a computer-readable form, allowing for more facile manipulation of large amounts of data; and, most importantly, (iv) use of fluorescent labels eliminates the problems associated with storage, use, and disposal of radioactive material.
Two classes of real-time multilane fluorescence detectors currently exist: (i) simultaneous detection systems which continuously excite and detect fluorescence from all electrophoresis lanes simultaneously, and, (ii) scanning detection systems which serially scan across the electrophoresis lanes, reading fluorescence one lane at a time.
While simultaneous-detection systems eliminate the need for moving parts, simultaneous-detection systems have several important practical drawbacks: (i) simultaneous detection systems require either large f-number collection optics or a large number of discrete independent detectors, each detector dedicated to continuously monitoring a single lane at a single color; and, (ii) simultaneous detection systems require a high-power light source to illuminate all lanes at once to the extent necessary to efficiently excite fluorescence. However, large f-number collection optics necessarily collect a large proportion of unwanted scattered excitation light, thereby reducing the signal-to-noise ratio of the data. And, when using a large number of electrophoresis lanes, each lane containing samples having multiple colors, the cost of dedicated detectors is prohibitive. Moreover, calibration of multiple dectectors presents formidable obstacles to truly quantitative performance, particularly when multiple detectors are used to interrogate a single separation lane. These drawbacks become particularly burdensome for systems employing a large number of lanes, fast electrophoresis, and multicolor detection methodologies.
Currently, scanning detection systems employ a linear scanning pattern; i.e., the detection system traverses the width of a planar array of electrophoresis lanes in a direction normal to the direction of sample migration, stopping and changing direction after each scan. As the speed of electrophoretic separations and throughput requirements increase, certain inherent limitations of these systems become apparent. First, because the scanner must decelerate, stop and change direction, and accelerate after each scan, the scanning system requires a high-power motor to effect a rapid acceleration and deceleration of the scanner. Furthermore, the electrophoresis lanes lying in the acceleration/deceleration zone are not useable because of the indeterminate scanner velocity as it transits these zones.
Another limitation of currently available linear scanners stems from imperfections in the scanning mechanisms themselves. Because of these imperfections, linear scanners invariably scan differently in one direction than the other. These differences between "forward" and "reverse" scans create significant ambiguities in data interpretation.
A third limitation of existing systems arises as a result of the planar geometry of the electrophoresis lanes and the linear scanning pattern. Because the time difference between the forward and reverse scans is smaller for lanes located at the edges of a planar array, the data collection frequency for a given lane is a function of the lane's position relative to the center of the array. This effect, known as "aliasing", can lead to an under-sampling of the lanes located near the edges of the array, thereby leading to nonuniform data quality across the lanes, particularly in the case of fast, high performance electrophoresis.
A fourth drawback to currently available scanners, particularly when applied to highly multiplexed arrays of separation lanes, is their low duty cycle per lane. As used herein, the term "duty-cycle" refers to the fraction of a scan cycle for which the optical detection system is looking at a given separation lane. Low duty cycle is particularly problematic in the case of fluorescence detection, where, in the absence of photodestruction of the fluorescent dyes and assuming a constant illumination intensity, the signal-to-noise ratio of the fluorescence signal is proportional to the square root of the time that the optical detection system interrogates the sample. Therefore, a reduced duty cycle means a lower fluorescent signal. The problem of low duty cycle is particularly acute for the case of capillary arrays because of the relatively thick walls typically used in capillary tubes, e.g., 25 .mu.m internal radius capillary will have a 170 .mu.m thick wall. Thus, in the case of capillary tubes, even when neighboring capillaries are touching each other, the maximum duty cycle will be low. Using current scanners, the only way to make up for a low duty cycle is by (i) limiting the number of separation lanes, thereby limiting the throughput of the system; or, (2) increasing the intensity of the light source, thereby greatly increasing the cost of the system.